Genotyping by next-generation sequencing

ABSTRACT

Provided herein is technology relating to genotyping and particularly, but not exclusively, to methods for genotyping one or more organisms by genome sequencing.

This application claims priority to U.S. provisional patent application61/586,596, filed Jan. 13, 2012, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant NumbersIOS-1027527, IOS-0820610, IOS-0910642, and DEB0919348 awarded by theNational Science Foundation. The government has certain rights in theinvention.

FIELD OF INVENTION

Provided herein is technology relating to genotyping and particularly,but not exclusively, to methods for genotyping one or more subjects bygenome sequencing.

BACKGROUND

Next-generation sequencing enables researchers to obtain large amountsof data at a reduced cost and thus provides a tremendous opportunity togenotype an individual of any species in depth (Lai et al. “Genome-widepatterns of genetic variation among elite maize inbred lines” 2010, NatGenet 42: 1027-1030). Recently, several genotyping-by-sequencing (GBS)approaches were developed to genotype hundreds of individualssimultaneously (Andolfatto et al. “Multiplexed shotgun genotyping forrapid and efficient genetic mapping” 2011, Genome Res 21: 610-17; Bairdet al. “Rapid SNP discovery and genetic mapping using sequenced RADmarkers” 2008, PLoS One 3: e3376; Elshire et al. “A Robust, SimpleGenotyping-by-Sequencing (GBS) Approach for High Diversity Species” 2011PLoS One 6: e19379).

Conventional genotyping is most often conducted using pre-defined SNPmarkers that must be discovered and validated in advance; these markersare often population-specific. These SNPs are typically detected viahybridization or by individual SNP-specific PCR-based assays. Incontrast, GBS technology enables the detection of a wider range ofpolymorphisms than PCR-based assays (e.g., SNPs plus small insertionsand/or deletions, e.g., “indels”). GBS technology eliminates the need topre-discover and validate polymorphisms. Hence, GBS can be used in anypolymorphic species and any segregating population.

However, conventional GBS methods share at least two drawbacks. First,conventional methods use double-stranded adaptors and, consequently,associated methods require stringent control of the template:adaptorconcentration ratio in the adaptor ligation. As a result, preciselyquantified, high quality input DNA is required as a starting material(see, e.g., Elshire et al.). Second, these methods survey hundreds ofthousands or more sites and thus require numerous sequencing reads togenerate enough coverage for each site in each sample.

SUMMARY

Accordingly, provided herein are technologies related to GBS. Inparticular, embodiments of the technology are provided that use asingle-stranded oligonucleotide in lieu of the conventionaldouble-stranded adaptors for the ligation reaction (see, e.g., Liu. etal. “DLA-based strategies for cloning insertion mutants: cloning the gl4locus of maize using Mu transposon tagged alleles” 2009 Genetics 183:1215-25, incorporated herein by reference in its entirety for allpurposes). For example, a single-stranded oligonucleotide is lesssubject to self-ligation as a double-stranded adaptor is and thus thetemplate:adaptor ratio is much less critical than in conventionalapproaches.

Furthermore, in some embodiments of the technology, a method ofsingle-stranded oligonucleotide digestion and ligation is used to“barcode” or index a nucleic acid (e.g., a DNA). Consequently, in someembodiments, numerous (e.g., hundreds, thousands, tens of thousands,millions, etc.) barcoded DNAs are combined into a single multiplexedsample for analysis while maintaining source information in the barcodefor each DNA. After analysis, the data are deconvoluted to obtain thedata relevant to each barcoded DNA.

In some embodiments of the technology, two restriction enzymes are usedto generate two sites with different overhangs at each end of thedigested fragments. One site is ligated with the barcode oligonucleotideto permit multiplexing of samples during analysis, e.g., sequencing. Theother site is ligated with an oligonucleotide without the barcode. Thenumber of sites targeted for analysis (e.g., by sequencing) is furtherreduced by the design and selection of amplification primerscomplementary to the non-barcode site. By manipulating the choice ofrestriction enzymes and barcode sequences embodiments of the GBStechnology provided herein are “tunable” in that a researcher is able toassay genotypes at a pre-defined number of genetic markers and multiplexthe genotyping of the desired number of individuals. For example, insome embodiments of the technology, hundreds of markers are assayed perindividual, while in other embodiments thousands or even tens ofthousands of markers are assayed per individual. If fewer markers areassayed, then less sequencing is needed per individual. Consequently,costs are reduced by multiplexing multiple samples in one experiment andgenotyping more individuals per unit of cost.

In some embodiments, a restriction enzyme is used that generates afragment having an overhang that is ligated with a barcodeoligonucleotide. The barcode oligonucleotide comprises a sequencecomplementary to the overhang, the DNA barcode, and a common sequenceused as, e.g., a primer binding site for amplification. Using differentbarcodes allows the pooling of DNAs from different sources and thesubsequent deconvolution of data for each individual subject providing aDNA. For a given sequence target of interest, a single primer isdesigned to bind to a region neighboring the enzyme recognition site atthe target. This single primer, combined with a second primercomplementary to the common sequence of the barcode oligonucleotide, isused for amplification, e.g., by PCR. In some embodiments, multipleprimers are designed in accord with the desired number of targets andassigned to a primer plex. In these embodiments, multiple targets areamplified in a pool of barcoded DNAs.

The technology finds use, e.g., to genotype a population with hundredsto thousands of individual subjects even in the absence of priorgenotyping information. The ability to tune the GBS technology providesresearchers with a unique flexibility to apply GBS to a wide variety ofprojects, e.g., in the seed and livestock breeding industries, forprotection of intellectual property, in the field of forensics, and forpaternity testing in both humans and livestock. This list is intended tobe exemplary and not limiting of the applications suitable for thetechnology provided. Moreover, some embodiments comprise targetenrichment methods (e.g., sequence capture) to sequence targeted regionson a large number of individuals. Additional embodiments andapplications will be apparent to persons skilled in the relevant artbased on the teachings contained herein.

Accordingly, provided herein are embodiments of methods comprisingdigesting a nucleic acid with a restriction enzyme to produce afragment; ligating a single-stranded barcode oligonucleotide to thefragment to produce a template; amplifying the template to produce anamplicon; and sequencing the amplicon to produce a sequence read. Insome embodiments, a template pool is produced by mixing a plurality oftemplates, e.g., in some embodiments, a template pool is produced bymixing a plurality of templates from a plurality of individuals. Someembodiments further provide parsing the sequence read, mapping thesequence read, and assigning a genotype. In some embodiments of themethods, the nucleic acid is digested with two different restrictionenzymes, e.g., NspI and BfuCI. Some embodiments provide that thesingle-stranded barcode oligonucleotide identifies a subject that wasthe source of the nucleic acid. Some embodiments provide that theamplifying comprises the use of a target specific primer, e.g., toselect an amplicon for sequencing.

As such, the technology described provides a method for genotyping bysequencing, the method comprising providing a first plurality of nucleicacids from a first subject; providing a second plurality of nucleicacids from a second subject; digesting the first plurality of nucleicacids with a restriction enzyme to produce a first plurality offragments; digesting the second plurality of nucleic acids with therestriction enzyme to produce a second plurality of fragments; ligatinga first single-stranded barcode oligonucleotide to each fragment of thefirst plurality of fragments to produce a first plurality of templates;ligating a second single-stranded barcode oligonucleotide to eachfragment of the second plurality of fragments to produce a secondplurality of templates; mixing the first plurality of templates and thesecond plurality of templates to produce a template pool; amplifying asubset of the template pool using a target specific primer to produce aplurality of amplicons; sequencing the plurality of amplicons to producea plurality of sequence reads; and deconvoluting the sequence readsusing a first sequence of the first barcode oligonucleotide and a secondsequence of the second barcode oligonucleotide.

Moreover, associated with the technology are embodiments of compositionscomprising a single-stranded barcode oligonucleotide, wherein thesingle-stranded barcode oligonucleotide comprises a sequence selectedfrom the group consisting of SEQ ID NOs: 1-23. In some embodiments, thecompositions further comprise a second single-stranded oligonucleotide.In some embodiments, a composition is provided comprising a nucleicacid, wherein the nucleic acid sequence comprises a sequence of asingle-stranded barcode oligonucleotide, a sequence of a target site,and a sequence of a second single-stranded oligonucleotide. Someembodiments of the compositions further comprise a first amplificationprimer complementary to the single-stranded barcode oligonucleotide anda target specific amplification primer complementary to the secondsingle-stranded oligonucleotide.

Moreover, the technology provides embodiments of a use of a compositiondescribed above for genotyping one or more subjects and embodiments of akit comprising a composition described above.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presenttechnology will become better understood with regard to the followingdrawings:

FIG. 1 is a drawing describing a method embodiment of the technologyprovided herein.

FIG. 2 is a drawing describing a method embodiment of the technologyprovided herein.

FIG. 3 is a drawing describing a method embodiment of the technologyprovided herein.

FIG. 4 is a drawing describing a method embodiment of the technologyprovided herein.

DETAILED DESCRIPTION

Provided herein is technology relating to genotyping and particularly,but not exclusively, to methods for genotyping one or more subjects bygenome sequencing. Is some embodiments, the technology usessingle-stranded barcode oligonucleotides and target selection to tunethe sequencing.

DEFINITIONS

To facilitate an understanding of the present technology, a number ofterms and phrases are defined below. Additional definitions are setforth throughout the detailed description.

Throughout the specification and claims, the following terms take themeanings explicitly associated herein, unless the context clearlydictates otherwise. The phrase “in one embodiment” as used herein doesnot necessarily refer to the same embodiment, though it may.Furthermore, the phrase “in another embodiment” as used herein does notnecessarily refer to a different embodiment, although it may. Thus, asdescribed below, various embodiments of the invention may be readilycombined, without departing from the scope or spirit of the invention.

In addition, as used herein, the term “or” is an inclusive “or” operatorand is equivalent to the term “and/or” unless the context clearlydictates otherwise. The term “based on” is not exclusive and allows forbeing based on additional factors not described, unless the contextclearly dictates otherwise. In addition, throughout the specification,the meaning of “a”, “an”, and “the” include plural references. Themeaning of “in” includes “in” and “on.”

The term “subject” refers to a biological organism such as a human orother animal (e.g., a pig, a cow, a mouse, etc.) and the like, or aplant, bacterium, archaon, or virus. In some embodiments, any entityhaving a genotype is a subject.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises coding sequences necessary for the production of an RNAor a polypeptide or its precursor. The term “portion” when used inreference to a gene refers to fragments of that gene. The fragments mayrange in size from a few nucleotides to the entire gene sequence minusone nucleotide. Thus, “a nucleotide comprising at least a portion of agene” may comprise fragments of the gene or the entire gene.

The term “gene” also encompasses the coding regions of a structural geneand includes sequences located adjacent to the coding region on both the5′ and 3′ ends for a distance of about 1 kb on either end such that thegene corresponds to the length of the full-length mRNA. The sequenceswhich are located 5′ of the coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the coding region and which are presenton the mRNA are referred to as 3′ non-translated sequences. The term“gene” encompasses both cDNA and genomic forms of a gene. A genomic formor clone of a gene contains the coding region interrupted withnon-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are segments of a gene which aretranscribed into nuclear RNA (hnRNA); introns may contain regulatoryelements such as enhancers. Introns are removed or “spliced out” fromthe nuclear or primary transcript; introns therefore are absent in themessenger RNA (mRNA) transcript. The mRNA functions during translationto specify the sequence or order of amino acids in a nascentpolypeptide.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequenceswhich are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers which control or influence thetranscription of the gene. The 3′ flanking region may contain sequenceswhich direct the termination of transcription, posttranscriptionalcleavage and polyadenylation.

The term “heterologous” when used in reference to a gene refers to agene encoding a factor that is not in its natural environment (i.e., hasbeen altered by the hand of man). For example, a heterologous geneincludes a gene from one species introduced into another species. Aheterologous gene also includes a gene native to an organism that hasbeen altered in some way (e.g., mutated, added in multiple copies,linked to a non-native promoter or enhancer sequence, etc.).Heterologous genes may comprise gene sequences that comprise cDNA formsof a gene; the cDNA sequences may be expressed in either a sense (toproduce mRNA) or anti-sense orientation (to produce an anti-sense RNAtranscript that is complementary to the mRNA transcript). Heterologousgenes are distinguished from endogenous genes in that the heterologousgene sequences are typically joined to nucleotide sequences comprisingregulatory elements such as promoters that are not found naturallyassociated with the gene for the protein encoded by the heterologousgene or with gene sequences in the chromosome, or are associated withportions of the chromosome not found in nature (e.g., genes expressed inloci where the gene is not normally expressed).

As used herein, the phrase “dNTP” means deoxynucleotidetriphosphate,where the nucleotide is any nucleotide, such as A, T, C, G or U.

As used herein, a “nucleic acid” shall mean any nucleic acid molecule,including, without limitation, DNA, RNA and hybrids thereof. The nucleicacid bases that form nucleic acid molecules can be the bases A, C, G, Tand U, as well as derivatives thereof. Derivatives of these bases arewell known in the art. The term should be understood to include, asequivalents, analogs of either DNA or RNA made from nucleotide analogs.The term as used herein also encompasses cDNA, that is complementary, orcopy, DNA produced from an RNA template, for example by the action ofreverse transcriptase.

The term “nucleotide sequence of interest” or “nucleic acid sequence ofinterest” refers to any nucleotide sequence (e.g., RNA or DNA), themanipulation of which may be deemed desirable for any reason (e.g., foranalysis, for quantification, to treat disease, confer improvedqualities, etc.) by one of ordinary skill in the art. Such nucleotidesequences include, but are not limited to, coding sequences ofstructural genes (e.g., reporter genes, selection marker genes,oncogenes, drug resistance genes, growth factors, etc.), and non-codingregulatory sequences which do not encode an mRNA or protein product(e.g., promoter sequence, polyadenylation sequence, terminationsequence, enhancer sequence, etc.).

The terms “oligonucleotide” or “polynucleotide” or “nucleotide” or“nucleic acid” refer to a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three, andusually more than ten. The exact size will depend on many factors, whichin turn depends on the ultimate function or use of the oligonucleotide.The oligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof. When present in a DNA form, the oligonucleotide may besingle-stranded (i.e., the sense strand) or double-stranded.

The terms “complementary” and “complementarity” refer to polynucleotides(e.g., a sequence of nucleotides) related by the base-pairing rules. Forexample, for the sequence 5′-A-G-T-3′ is complementary to the sequence3′-T-C-A-5′. Complementarity may be “partial,” in which only some of thenucleic acids' bases are matched according to the base pairing rules.Or, there may be “complete” or “total” complementarity between thenucleic acids. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methodswhich depend upon binding between nucleic acids.

The term “wild-type” when made in reference to a gene refers to a genethat has the characteristics of a gene isolated from a naturallyoccurring source. The term “wild-type” when made in reference to a geneproduct refers to a gene product that has the characteristics of a geneproduct isolated from a naturally occurring source. The term“naturally-occurring” as applied to an object refers to the fact that anobject can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory isnaturally-occurring. A wild-type gene is frequently that gene which ismost frequently observed in a population and is thus arbitrarilydesignated the “normal” or “wild-type” form of the gene. In contrast,the term “modified” or “mutant” when made in reference to a gene or to agene product refers, respectively, to a gene or to a gene product whichdisplays modifications in sequence and/or functional properties (i.e.,altered characteristics) when compared to the wild-type gene or geneproduct. It is noted that naturally-occurring mutants can be isolated;these are identified by the fact that they have altered characteristicswhen compared to the wild-type gene or gene product.

As used herein, an “allele” refers to an alternative sequence at aparticular locus; the length of an allele can be as small as 1nucleotide base, but is typically larger. Allelic sequence can be aminoacid sequence or nucleic acid sequence.

As used herein, a “locus” is a short sequence that is usually unique andusually found at one particular location in the genome by a point ofreference; e.g., a short DNA sequence that is a gene, or part of a geneor intergenic region. In some embodiments, a locus is a unique PCRproduct at a particular location in the genome. Loci may comprise one ormore polymorphisms; i.e., alternative alleles present in someindividuals.

Thus, the terms “variant” and “mutant” when used in reference to anucleotide sequence refer to an nucleic acid sequence that differs byone or more nucleotides from another, usually related nucleotide acidsequence. A “variation” is a difference between two different nucleotidesequences; typically, one sequence is a reference sequence.

As used herein, “marker” means a polymorphic nucleic acid sequence ornucleic acid feature. In a broader aspect, a “marker” can be adetectable characteristic that can be used to discriminate betweenheritable differences between organisms. Examples of suchcharacteristics may include genetic markers, protein composition,protein levels, oil composition, oil levels, carbohydrate composition,carbohydrate levels, fatty acid composition, fatty acid levels, aminoacid composition, amino acid levels, biopolymers, pharmaceuticals,starch composition, starch levels, fermentable starch, fermentationyield, fermentation efficiency, energy yield, secondary compounds,metabolites, morphological characteristics, and agronomiccharacteristics.

As used herein, “polymorphism” means the presence of one or morevariations of a nucleic acid sequence at one or more loci in apopulation of one or more individuals. The variation may comprise but isnot limited to one or more base changes, the insertion of one or morenucleotides or the deletion of one or more nucleotides. A polymorphismincludes a single nucleotide polymorphism (SNP), a simple sequencerepeat (SSR) and indels, which are insertions and deletions. Apolymorphism may arise from random processes in nucleic acidreplication, through mutagenesis, as a result of mobile genomicelements, from copy number variation and during the process of meiosis,such as unequal crossing over, genome duplication and chromosome breaksand fusions. The variation can be commonly found or may exist at lowfrequency within a population, the former having greater utility ingeneral plant breeding and the later may be associated with rare butimportant phenotypic variation. In some embodiments, a “polymorphism” isa variation among individuals in sequence, particularly in DNA sequence,or feature, such as a transcriptional profile or methylation pattern.Useful polymorphisms include single nucleotide polymorphisms (SNPs),insertions or deletions in DNA sequence (indels), simple sequencerepeats of DNA sequence (SSRs) a restriction fragment lengthpolymorphism, a haplotype, and a tag SNP. A genetic marker, a gene, aDNA-derived sequence, a RNA-derived sequence, a promoter, a 5′untranslated region of a gene, a 3′ untranslated region of a gene,microRNA, siRNA, a QTL, a satellite marker, a transgene, mRNA, ds mRNA,a transcriptional profile, and a methylation pattern may comprisepolymorphisms.

The term “polymorphic locus” refers to a genetic locus present in apopulation that shows variation between members of the population.

The term “detection assay” refers to an assay for detecting the presenceor absence of a wild-type or variant nucleic acid sequence (e.g.,mutation or polymorphism) in a given allele of a particular gene, or fordetecting the presence or absence of a particular protein or theactivity or effect of a particular protein or for detecting the presenceor absence of a variant of a particular protein.

As used herein, “typing” refers to any method whereby the specificallelic form of a given corn genomic polymorphism is determined. Forexample, a single nucleotide polymorphism (SNP) is typed by determiningwhich nucleotide is present (e.g., an A, G, T, or C).Insertion/deletions (indels) are determined by determining if the indelis present. Indels can be typed by a variety of assays including, butnot limited to, marker assays.

As used herein, the term “single nucleotide polymorphism,” also referredto by the abbreviation “SNP,” means a polymorphism at a single sitewherein the polymorphism constitutes a single base pair change, aninsertion of one or more base pairs, or a deletion of one or more basepairs.

As used herein, “genotype” means the genetic component of the phenotypeand it can be indirectly characterized using markers or directlycharacterized by nucleic acid sequencing. Suitable markers include aphenotypic character, a metabolic profile, a genetic marker, or someother type of marker. A genotype may constitute an allele for at leastone genetic marker locus or a haplotype for at least one haplotypewindow. In some embodiments, a genotype may represent a single locus andin others it may represent a genome-wide set of loci. In anotherembodiment, the genotype can reflect the sequence of a portion of achromosome, an entire chromosome, a portion of the genome, and theentire genome.

As used herein, “phenotype” means the detectable characteristics of acell or organism which are a manifestation of gene expression.

As used herein, “barcode” shall generally mean a virtual or a knownnucleotide sequence that is used as an index for labeling a DNA fragmentand/or a library and for constructing a multiplex library. A libraryincludes, but is not limited to, a genomic DNA library, a cDNA library,and a ChIP library. A plurality of DNAs, each of which is separatelylabeled with a distinct barcode, may be pooled together to form amultiplex barcoded library for performing sequencing simultaneously, inwhich each barcode is sequenced together with its flanking tags locatedin the same construct and thereby serves as a index for the DNA fragmentand/or library labeled by it. In some embodiments, a barcode is madewith a specific nucleotide sequence having 1, 2, 3, 4, 5, 6, or morenucleotides in length. The length of a barcode may be increased alongwith the maximum sequencing length of a sequencer. The terms “barcodedadaptor” and “barcoded adaptor sequence” are interchangeable. The terms“barcode” and “barcode sequence” are interchangeable.

As used herein, “virtual” shall generally mean not in actual form butexisting or resulting in effect.

As used herein, “index” shall generally mean a distinctive oridentifying mark or characteristic.

As used herein, “restriction enzyme recognition site” and “restrictionenzyme binding site” are interchangeable.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

The term “primer” refers to an oligonucleotide, whether occurringnaturally as in a purified restriction digest or produced synthetically,that is capable of acting as a point of initiation of synthesis whenplaced under conditions in which synthesis of a primer extension productwhich is complementary to a nucleic acid strand is induced, (i.e., inthe presence of nucleotides and an inducing agent such as DNA polymeraseand at a suitable temperature and pH). The primer is preferably singlestranded for maximum efficiency in amplification, but may alternativelybe double stranded. If double stranded, the primer is first treated toseparate its strands before being used to prepare extension products.Preferably, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. The exact lengths of the primers willdepend on many factors, including temperature, source of primer and theuse of the method.

One with ordinary skill in the art of design of primers will recognizethat a given primer need not hybridize with 100% complementarity toprime the synthesis of a complementary nucleic acid strand. Primer pairsequences may be a “best fit” amongst several aligned sequences, thusthey need not be fully complementary to the hybridization region of anyone of the sequences in the alignment. Moreover, a primer may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., for example, a loopstructure or a hairpin structure). The primers may comprise at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95% or at least 99% sequence identity with a target nucleic acid ofinterest. Thus, in some embodiments, an extent of variation of 70% to100%, or any range falling within, of the sequence identity is possiblerelative to the specific primer sequences disclosed herein. Toillustrate, determination of sequence identity is described in thefollowing example: a primer 20 nucleobases in length which is identicalto another 20 nucleobase primer having two non-identical residues has 18of 20 identical residues (18/20=0.9 or 90% sequence identity). Inanother example, a primer 15 nucleobases in length having all residuesidentical to a 15 nucleobase segment of primer 20 nucleobases in lengthwould have 15/20=0.75 or 75% sequence identity with the 20 nucleobaseprimer. Percent identity need not be a whole number, for example when a28 consecutive nucleobase primer is completely identical to a 31consecutive nucleobase primer (28/31=0.9032 or 90.3% identical).

Percent homology, sequence identity or complementarity, can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Insome embodiments, complementarity of primers with respect to theconserved priming regions of viral nucleic acid, is between about 70%and about 80%. In other embodiments, homology, sequence identity orcomplementarity, is between about 80% and about 90%. In yet otherembodiments, homology, sequence identity or complementarity, is at least90%, at least 92%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or is 100%.

In some embodiments, the primers described herein comprise at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, atleast 94%, at least 95%, at least 96%, at least 98%, or at least 99%, or100% (or any range falling within) sequence identity with the primersequences specifically disclosed herein.

In some embodiments, any given primer comprises a modificationcomprising the addition of a non-templated T residue to the 5′ end ofthe primer (i.e., the added T residue does not necessarily hybridize tothe nucleic acid being amplified). The addition of a non-templated Tresidue has an effect of minimizing the addition of non-templated Aresidues as a result of the non-specific enzyme activity of, e.g., TaqDNA polymerase (Magnuson et al., Biotechniques, 1996: 21, 700-709), anoccurrence which may lead to ambiguous results arising from molecularmass analysis.

Primers may contain one or more universal bases. Because any variation(due to codon wobble in the third position) in the conserved regionsamong species is likely to occur in the third position of a DNA (or RNA)triplet, oligonucleotide primers can be designed such that thenucleotide corresponding to this position is a base which can bind tomore than one nucleotide, referred to herein as a “universalnucleobase.” For example, under this “wobble” base pairing, inosine (I)binds to U, C or A; guanine (G) binds to U or C, and uridine (U) bindsto U or C. Other examples of universal nucleobases include nitroindolessuch as 5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides andNucleotides, 1995, 14, 1001-1003), the degenerate nucleotides dP or dK,an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot etal., Nucleosides and Nucleotides., 1995, 14, 1053-1056) or the purineanalog 1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide (Sala etal., Nucl. Acids Res., 1996, 24, 3302-3306).

In some embodiments, to compensate for weaker binding by the wobblebase, oligonucleotide primers are configured such that the first andsecond positions of each triplet are occupied by nucleotide analogs thatbind with greater affinity than the unmodified nucleotide. Examples ofthese analogs include, but are not limited to, 2,6-diaminopurine whichbinds to thymine, 5-propynyluracil which binds to adenine and5-propynylcytosine and phenoxazines, including G-clamp, which binds toG. Propynylated pyrimidines are described in U.S. Pat. Nos. 5,645,985,5,830,653 and 5,484,908, incorporated herein by reference in theirentireties. Propynylated primers are described in U.S. Pat. Appl. Pub.No. 2003-0170682, incorporated herein by reference in its entirety.Phenoxazines are described in U.S. Pat. Nos. 5,502,177, 5,763,588, and6,005,096, each of which is incorporated herein by reference in itsentirety. G-clamps are described in U.S. Pat. Nos. 6,007,992 and6,028,183, each of which is incorporated herein by reference in itsentirety.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” refers to a nucleic acid sequence that isidentified and separated from at least one contaminant nucleic acid withwhich it is ordinarily associated in its natural source. Isolatednucleic acid is present in a form or setting that is different from thatin which it is found in nature. In contrast, non-isolated nucleic acids,such as DNA and RNA, are found in the state they exist in nature.Examples of non-isolated nucleic acids include: a given DNA sequence(e.g., a gene) found on the host cell chromosome in proximity toneighboring genes; RNA sequences, such as a specific mRNA sequenceencoding a specific protein, found in the cell as a mixture withnumerous other mRNAs which encode a multitude of proteins. However,isolated nucleic acid encoding a particular protein includes, by way ofexample, such nucleic acid in cells ordinarily expressing the protein,where the nucleic acid is in a chromosomal location different from thatof natural cells, or is otherwise flanked by a different nucleic acidsequence than that found in nature. The isolated nucleic acid oroligonucleotide may be present in single-stranded or double-strandedform. When an isolated nucleic acid or oligonucleotide is to be utilizedto express a protein, the oligonucleotide will contain at a minimum thesense or coding strand (i.e., the oligonucleotide may single-stranded),but may contain both the sense and anti-sense strands (i.e., theoligonucleotide may be double-stranded).

The term “purified” refers to molecules, either nucleic or amino acidsequences, that are removed from their natural environment, isolated orseparated. An “isolated nucleic acid sequence” may therefore be apurified nucleic acid sequence. “Substantially purified” molecules areat least 60% free, preferably at least 75% free, and more preferably atleast 90% free from other components with which they are naturallyassociated. As used herein, the term “purified” or “to purify” alsorefer to the removal of contaminants from a sample. The removal ofcontaminating proteins results in an increase in the percent ofpolypeptide of interest in the sample. In another example, recombinantpolypeptides are expressed in plant, bacterial, yeast, or mammalian hostcells and the polypeptides are purified by the removal of host cellproteins; the percent of recombinant polypeptides is thereby increasedin the sample.

The term “composition comprising” a given polynucleotide sequence refersbroadly to any composition containing the given polynucleotide sequence.The composition may comprise an aqueous solution, e.g., containing salts(e.g., NaCl), detergents (e.g., SDS), and other components.

The term “sample” is used in its broadest sense. In one sense it canrefer to an animal cell or tissue. In another sense, it is meant toinclude a specimen or culture obtained from any source, as well asbiological and environmental samples. Biological samples may be obtainedfrom plants or animals (including humans) and encompass fluids, solids,tissues, and gases. Environmental samples include environmental materialsuch as surface matter, soil, water, and industrial samples. Theseexamples are not to be construed as limiting the sample types applicableto the present invention.

Embodiments of the Technology

In one aspect, the technology provides methods forgenotyping-by-sequencing. For example, in some embodiments, a pluralityof DNAs is sequenced at one or more loci, markers, SNPs, or other sitesof interest. The methods provide that DNA is digested with a restrictionenzyme, e.g., a sample comprising 10 ng, 100 ng, 1000 ng, etc. ofgenomic DNA (in some embodiments, RNase treated DNA) is digested withone or more restriction enzymes, e.g., NspI and/or BfuCI, in anappropriate buffer (e.g., a commercially available buffer (e.g., assupplied by NEB)) under appropriate conditions for digestion. Afterdigestion the digested fragments are ligated to a single-strandedbarcoded (indexed) oligonucleotide, e.g., as provided in Table 1. Insome embodiments, a second oligonucleotide, e.g., as provided in Table2, is ligated to the other end of the digested fragments.

TABLE 1 Barcode oligonucleotide sequences SEQ NO: name sequence ID nspl15′ ACACGACGCTCTTCCGATCTCGTATATGCATG 3′  1 nspl2 5′ACACGACGCTCTTCCGATCTCACGCTACATG 3′  2 nspl3 5′ACACGACGCTCTTCCGATCTCCGAGTGACATG 3′  3 nspl4 5′ACACGACGCTCTTCCGATCTCGTACTGTCATG 3′  4 nspl5 5′ACACGACGCTCTTCCGATCTATGTGCTACATG 3′  5 nspl6 5′ACACGACGCTCTTCCGATCTGGTCTCACCATG 3′  6 nspl7 5′ACACGACGCTCTTCCGATCTAGACTCGCATG 3′  7 nspl8 5′ACACGACGCTCTTCCGATCTATACTCGCCATG 3′  8 nspl9 5′ACACGACGCTCTTCCGATCTATGAGACCATG 3′  9 nspl10 5′ACACGACGCTCTTCCGATCTACTCGATACATG 3′ 10 nspl12 5′ACACGACGCTCTTCCGATCTGCTAGTAGCATG 3′ 11 nspl13 5′ACACGACGCTCTTCCGATCTCTGCGAGTCATG 3′ 12 nspl14 5′ACACGACGCTCTTCCGATCTGGCTACTGCATG 3′ 13 nspl15 5′ACACGACGCTCTTCCGATCTACGCATGTCATG 3′ 14 nspl16 5′ACACGACGCTCTTCCGATCTCATCTACTCATG 3′ 15 nspl18 5′ACACGACGCTCTTCCGATCTGAGACACACATG 3′ 16 nspl19 5′ACACGACGCTCTTCCGATCTCAGCGTACCATG 3′ 17 nspl21 5′ACACGACGCTCTTCCGATCTGCTCTACACATG 3′ 18 nspl22 5′ACACGACGCTCTTCCGATCTCCTGCATACATG 3′ 19 nspl23 5′ACACGACGCTCTTCCGATCTACACTGATCATG 3′ 20 nspl97 5′ACACGACGCTCTTCCGATCTCATAGCTGCATG 3′ 21 nspl99 5′ACACGACGCTCTTCCGATCTCGTACTACATG 3′ 22 nspl100 5′ACACGACGCTCTTCCGATCTGATATGTGCATG 3′ 23

The barcode oligonucleotide comprises a sequence common to every barcodeoligonucleotide, a barcode sequence that is unique to every barcodeoligonucleotide, and a sequence that is complementary to thesingle-stranded end produced by the restriction enzyme. In someembodiments, the second oligonucleotide comprises a sequence that iscomplementary to a single-stranded end produced by the secondrestriction enzyme or, in some embodiments, to a sequence near oradjacent to a target site of interest (e.g., a marker, SNP, allele,locus, polymorphic site, etc.). In some embodiments the barcodeoligonucleotide comprises a phosphorothioate linkage, e.g., after the 5′A in the sequences provided in Table 1.

TABLE 2 Second (non-barcode) oligonucleotide sequences name 5′sequence 3′ SEQ ID NO: bfuci12mm 5′ GATCTGAAGAGCTCGT 3′ 24 s-SEO15 5′P-GATCGGAAGAGCTC*G 3′ 29 An asterisk (*) denotes a phosphorothioate bondand a P- denotes 5′-phosphorylation

In some embodiments, multiple ligated samples (e.g., from multiplesubjects, samples, sources, BACs, etc.) are mixed to provide a pooledsample. In some embodiments, the samples are purified to removecontaminants or components from previous reactions (e.g., salts,enzymes) that may inhibit subsequent steps of the methods. In someembodiments, the purification is performed using a commercial kit, e.g.,the Qiaquick PCR purification kit (Qiagen, Cat#28106 or Cat#28104). Insome embodiments, the sample is size-selected, e.g., to enrich thesample for fragments greater than 250 bp in size, e.g., using AMPurebeads (Agencourt, Beckman Coulter).

In some embodiments, DNAs in the pooled sample comprise multiplemarkers, SNPs, loci, target sites, BACs, etc. Accordingly, in someembodiments an amplification (e.g., PCR) using one or more targetselection primers in combination with a common primer selects one ormore target sites for further analysis (e.g., by specifically enrichingtarget sites in the sample DNAs). Amplification primers comprise aphosphorothioate bond in some embodiments.

In some embodiments, nucleic acid molecules are analyzed andcharacterized by any of a wide variety of methods, including, but notlimited to, sequencing, hybridization analysis, amplification (e.g., viapolymerase chain reaction (PCR), reverse transcription polymerase chainreaction (RT-PCR), transcription-mediated amplification (TMA), ligasechain reaction (LCR), strand displacement amplification (SDA), andnucleic acid sequence based amplification (NASBA)).

Finally, in some embodiments, the sample is subjected to anotheramplification to produce a sample suitable for sequencing, e.g., byusing primer 1.1 and final3 as shown in Table 3. Amplification primerscomprise a phosphorothioate bond in some embodiments.

TABLE 3 amplification oligonucleotides SEQ ID name sequence (5′ to 3′)NO: bfuci3ACAA ACGAGCTCTTCCGATCTGTT 25 common ACACGACGCTCTTCCGATCT 26s-common1 A*CACGACGCTCTTCCGATC*T 33 s-SEO16CTA G*A*T*CTAGCCTTCTCGAGCA 30primer 1.1 AATGATACGGCGACCACCGAGATCTACACTCT 27TTCCCTACACGACGCTCTTCCGATCT final3 CAAGCAGAAGACGGCATACGAGCTCTTCCGAT 28CTGT s-SE-P1 A*ATGATACGGCGACCACCGAGATCTACACTC 31TTTCCCTACACGACGCTCTTCCGATC*T s-SE-P2AG C*AAGCAGAAGACGGCATACGAGCTCTTCCGA32 TCTA*G An asterisk (*) denotes a phosphorothioate bond

In some embodiments, the technology employs a sequencing technology. Insome aspects, DNA sequencing methodologies associated with the presenttechnology comprise Second Generation (a.k.a. Next Generation orNext-Gen), Third Generation (a.k.a. Next-Next-Gen), or Fourth Generation(a.k.a. N3-Gen) sequencing technologies including, but not limited to,pyrosequencing, sequencing-by-ligation, single molecule sequencing,sequence-by-synthesis (SBS), massive parallel clonal, massive parallelsingle molecule SBS, massive parallel single molecule real-time, massiveparallel single molecule real-time nanopore technology, etc. Morozovaand Marra provide a review of some such technologies in Genomics, 92:255 (2008), herein incorporated by reference in its entirety. Those ofordinary skill in the art will recognize that because RNA is less stablein the cell and more prone to nuclease attack experimentally RNA isusually reverse transcribed to DNA before sequencing.

A number of DNA sequencing techniques are known in the art, includingfluorescence-based sequencing methodologies (See, e.g., Birren et al.,Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N.Y.; hereinincorporated by reference in its entirety). In some embodiments,automated sequencing techniques understood in that art are utilized. Insome embodiments, the present technology provides parallel sequencing ofpartitioned amplicons (PCT Publication No: WO2006084132 to KevinMcKernan et al., herein incorporated by reference in its entirety). Insome embodiments, DNA sequencing is achieved by parallel oligonucleotideextension (See, e.g., U.S. Pat. No. 5,750,341 to Macevicz et al., andU.S. Pat. No. 6,306,597 to Macevicz et al., both of which are hereinincorporated by reference in their entireties). Additional examples ofsequencing techniques include the Church polony technology (Mitra etal., 2003, Analytical Biochemistry 320, 55-65; Shendure et al., 2005Science 309, 1728-1732; U.S. Pat. No. 6,432,360, U.S. Pat. No.6,485,944, U.S. Pat. No. 6,511,803; herein incorporated by reference intheir entireties), the 454 picotiter pyrosequencing technology(Margulies et al., 2005 Nature 437, 376-380; US 20050130173; hereinincorporated by reference in their entireties), the Solexa single baseaddition technology (Bennett et al., 2005, Pharmacogenomics, 6, 373-382;U.S. Pat. No. 6,787,308; U.S. Pat. No. 6,833,246; herein incorporated byreference in their entireties), the Lynx massively parallel signaturesequencing technology (Brenner et al. (2000). Nat. Biotechnol.18:630-634; U.S. Pat. No. 5,695,934; U.S. Pat. No. 5,714,330; hereinincorporated by reference in their entireties), and the Adessi PCRcolony technology (Adessi et al. (2000). Nucleic Acid Res. 28, E87; WO00018957; herein incorporated by reference in its entirety).

Next-generation sequencing (NGS) methods share the common feature ofmassively parallel, high-throughput strategies, with the goal of lowercosts in comparison to older sequencing methods (see, e.g., Voelkerdinget al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.Microbial., 7:287-296; each herein incorporated by reference in theirentirety). NGS methods can be broadly divided into those that typicallyuse template amplification and those that do not.Amplification-requiring methods include pyrosequencing commercialized byRoche as the 454 technology platforms (e.g., GS 20 and GS FLX), theSolexa platform commercialized by Illumina, and the SupportedOligonucleotide Ligation and Detection (SOLiD) platform commercializedby Applied Biosystems. Non-amplification approaches, also known assingle-molecule sequencing, are exemplified by the HeliScope platformcommercialized by Helicos BioSciences, and emerging platformscommercialized by VisiGen, Oxford Nanopore Technologies Ltd., LifeTechnologies/Ion Torrent, and Pacific Biosciences, respectively.

In pyrosequencing (Voelkerding et al., Clinical Chem., 55: 641-658,2009; MacLean et al., Nature Rev. Microbial., 7: 287-296; U.S. Pat. No.6,210,891; U.S. Pat. No. 6,258,568; each herein incorporated byreference in its entirety), template DNA is fragmented, end-repaired,ligated to adaptors, and clonally amplified in-situ by capturing singletemplate molecules with beads bearing oligonucleotides complementary tothe adaptors. Each bead bearing a single template type iscompartmentalized into a water-in-oil microvesicle, and the template isclonally amplified using a technique referred to as emulsion PCR. Theemulsion is disrupted after amplification and beads are deposited intoindividual wells of a picotitre plate functioning as a flow cell duringthe sequencing reactions. Ordered, iterative introduction of each of thefour dNTP reagents occurs in the flow cell in the presence of sequencingenzymes and luminescent reporter such as luciferase. In the event thatan appropriate dNTP is added to the 3′ end of the sequencing primer, theresulting production of ATP causes a burst of luminescence within thewell, which is recorded using a CCD camera. It is possible to achieveread lengths greater than or equal to 400 bases, and 10⁶ sequence readscan be achieved, resulting in up to 500 million base pairs (Mb) ofsequence.

In the Solexa/Illumina platform (Voelkerding et al., Clinical Chem., 55.641-658, 2009; MacLean et al., Nature Rev. Microbial., 7:287-296; U.S.Pat. No. 6,833,246; U.S. Pat. No. 7,115,400; U.S. Pat. No. 6,969,488;each herein incorporated by reference in its entirety), sequencing dataare produced in the form of shorter-length reads. In this method,single-stranded fragmented DNA is end-repaired to generate5′-phosphorylated blunt ends, followed by Klenow-mediated addition of asingle A base to the 3′ end of the fragments. A-addition facilitatesaddition of T-overhang adaptor oligonucleotides, which are subsequentlyused to capture the template-adaptor molecules on the surface of a flowcell that is studded with oligonucleotide anchors. The anchor is used asa PCR primer, but because of the length of the template and itsproximity to other nearby anchor oligonucleotides, extension by PCRresults in the “arching over” of the molecule to hybridize with anadjacent anchor oligonucleotide to form a bridge structure on thesurface of the flow cell. These loops of DNA are denatured and cleaved.Forward strands are then sequenced with reversible dye terminators. Thesequence of incorporated nucleotides is determined by detection ofpost-incorporation fluorescence, with each fluor and block removed priorto the next cycle of dNTP addition. Sequence read length ranges from 36nucleotides to over 50 nucleotides, with overall output exceeding 1billion nucleotide pairs per analytical run.

Sequencing nucleic acid molecules using SOLiD technology (Voelkerding etal., Clinical Chem., 55:641-658, 2009; MacLean et al., Nature Rev.Microbial., 7:287-296; U.S. Pat. No. 5,912,148; U.S. Pat. No. 6,130,073;each herein incorporated by reference in their entirety) also involvesfragmentation of the template, ligation to oligonucleotide adaptors,attachment to beads, and clonal amplification by emulsion PCR. Followingthis, beads bearing template are immobilized on a derivatized surface ofa glass flow-cell, and a primer complementary to the adaptoroligonucleotide is annealed. However, rather than utilizing this primerfor 3′ extension, it is instead used to provide a 5′ phosphate group forligation to interrogation probes containing two probe-specific basesfollowed by 6 degenerate bases and one of four fluorescent labels. Inthe SOLiD system, interrogation probes have 16 possible combinations ofthe two bases at the 3′ end of each probe, and one of four fluors at the5′ end. Fluor color, and thus identity of each probe, corresponds tospecified color-space coding schemes. Multiple rounds (usually 7) ofprobe annealing, ligation, and fluor detection are followed bydenaturation, and then a second round of sequencing using a primer thatis offset by one base relative to the initial primer. In this manner,the template sequence can be computationally re-constructed, andtemplate bases are interrogated twice, resulting in increased accuracy.Sequence read length averages 35 nucleotides, and overall output exceeds4 billion bases per sequencing run.

In certain embodiments, nanopore sequencing is employed (see, e.g.,Astier et al., J. Am. Chem. Soc. 2006 Feb. 8; 128(5)1705-10, hereinincorporated by reference). The theory behind nanopore sequencing has todo with what occurs when a nanopore is immersed in a conducting fluidand a potential (voltage) is applied across it. Under these conditions aslight electric current due to conduction of ions through the nanoporecan be observed, and the amount of current is exceedingly sensitive tothe size of the nanopore. As each base of a nucleic acid passes throughthe nanopore, this causes a change in the magnitude of the currentthrough the nanopore that is distinct for each of the four bases,thereby allowing the sequence of the DNA molecule to be determined.

In certain embodiments, HeliScope by Helicos BioSciences is employed(Voelkerding et al., Clinical Chem., 55. 641-658, 2009; MacLean et al.,Nature Rev. Microbiol., 7:287-296; U.S. Pat. No. 7,169,560; U.S. Pat.No. 7,282,337; U.S. Pat. No. 7,482,120; U.S. Pat. No. 7,501,245; U.S.Pat. No. 6,818,395; U.S. Pat. No. 6,911,345; U.S. Pat. No. 7,501,245;each herein incorporated by reference in their entirety). Template DNAis fragmented and polyadenylated at the 3′ end, with the final adenosinebearing a fluorescent label. Denatured polyadenylated template fragmentsare ligated to poly(dT) oligonucleotides on the surface of a flow cell.Initial physical locations of captured template molecules are recordedby a CCD camera, and then label is cleaved and washed away. Sequencingis achieved by addition of polymerase and serial addition offluorescently-labeled dNTP reagents. Incorporation events result influor signal corresponding to the dNTP, and signal is captured by a CCDcamera before each round of dNTP addition. Sequence read length rangesfrom 25-50 nucleotides, with overall output exceeding 1 billionnucleotide pairs per analytical run.

The Ion Torrent technology is a method of DNA sequencing based on thedetection of hydrogen ions that are released during the polymerizationof DNA (see, e.g., Science 327(5970): 1190 (2010); U.S. Pat. Appl. Pub.Nos. 20090026082, 20090127589, 20100301398, 20100197507, 20100188073,and 20100137143, incorporated by reference in their entireties for allpurposes). A microwell contains a template DNA strand to be sequenced.Beneath the layer of microwells is a hypersensitive ISFET ion sensor.All layers are contained within a CMOS semiconductor chip, similar tothat used in the electronics industry. When a dNTP is incorporated intothe growing complementary strand a hydrogen ion is released, whichtriggers a hypersensitive ion sensor. If homopolymer repeats are presentin the template sequence, multiple dNTP molecules will be incorporatedin a single cycle. This leads to a corresponding number of releasedhydrogens and a proportionally higher electronic signal. This technologydiffers from other sequencing technologies in that no modifiednucleotides or optics are used. The per base accuracy of the Ion Torrentsequencer is ˜99.6% for 50 base reads, with ˜100 Mb generated per run.The read-length is 100 base pairs. The accuracy for homopolymer repeatsof 5 repeats in length is ˜98%. The benefits of ion semiconductorsequencing are rapid sequencing speed and low upfront and operatingcosts. However, the cost of acquiring a pH-mediated sequencer isapproximately $50,000, excluding sample preparation equipment and aserver for data analysis.

Another exemplary nucleic acid sequencing approach that may be adaptedfor use with the present invention was developed by Stratos Genomics,Inc. and involves the use of Xpandomers. This sequencing processtypically includes providing a daughter strand produced by atemplate-directed synthesis. The daughter strand generally includes aplurality of subunits coupled in a sequence corresponding to acontiguous nucleotide sequence of all or a portion of a target nucleicacid in which the individual subunits comprise a tether, at least oneprobe or nucleobase residue, and at least one selectively cleavablebond. The selectively cleavable bond(s) is/are cleaved to yield anXpandomer of a length longer than the plurality of the subunits of thedaughter strand. The Xpandomer typically includes the tethers andreporter elements for parsing genetic information in a sequencecorresponding to the contiguous nucleotide sequence of all or a portionof the target nucleic acid. Reporter elements of the Xpandomer are thendetected. Additional details relating to Xpandomer-based approaches aredescribed in, for example, U.S. Pat. Pub No. 20090035777, entitled “HIGHTHROUGHPUT NUCLEIC ACID SEQUENCING BY EXPANSION,” filed Jun. 19, 2008,which is incorporated herein in its entirety.

Other emerging single molecule sequencing methods include real-timesequencing by synthesis using a VisiGen platform (Voelkerding et al.,Clinical Chem., 55: 641-58, 2009; U.S. Pat. No. 7,329,492; U.S. patentapplication Ser. No. 11/671,956; U.S. patent application Ser. No.11/781,166; each herein incorporated by reference in their entirety) inwhich immobilized, primed DNA template is subjected to strand extensionusing a fluorescently-modified polymerase and florescent acceptormolecules, resulting in detectible fluorescence resonance energytransfer (FRET) upon nucleotide addition.

Another real-time single molecule sequencing system developed by PacificBiosciences (Voelkerding et al., Clinical Chem., 55. 641-658, 2009;MacLean et al., Nature Rev. Microbiol., 7:287-296; U.S. Pat. No.7,170,050; U.S. Pat. No. 7,302,146; U.S. Pat. No. 7,313,308; U.S. Pat.No. 7,476,503; all of which are herein incorporated by reference)utilizes reaction wells 50-100 nm in diameter and encompassing areaction volume of approximately 20 zeptoliters (10⁻²¹ L). Sequencingreactions are performed using immobilized template, modified phi29 DNApolymerase, and high local concentrations of fluorescently labeleddNTPs. High local concentrations and continuous reaction conditionsallow incorporation events to be captured in real time by fluor signaldetection using laser excitation, an optical waveguide, and a CCDcamera.

In certain embodiments, the single molecule real time (SMRT) DNAsequencing methods using zero-mode waveguides (ZMWs) developed byPacific Biosciences, or similar methods, are employed. With thistechnology, DNA sequencing is performed on SMRT chips, each containingthousands of zero-mode waveguides (ZMWs). A ZMW is a hole, tens ofnanometers in diameter, fabricated in a 100 nm metal film deposited on asilicon dioxide substrate. Each ZMW becomes a nanophotonic visualizationchamber providing a detection volume of just 20 zeptoliters (10⁻²¹ L).At this volume, the activity of a single molecule can be detectedamongst a background of thousands of labeled nucleotides. The ZMWprovides a window for watching DNA polymerase as it performs sequencingby synthesis. Within each chamber, a single DNA polymerase molecule isattached to the bottom surface such that it permanently resides withinthe detection volume. Phospholinked nucleotides, each type labeled witha different colored fluorophore, are then introduced into the reactionsolution at high concentrations which promote enzyme speed, accuracy,and processivity. Due to the small size of the ZMW, even at these high,biologically relevant concentrations, the detection volume is occupiedby nucleotides only a small fraction of the time. In addition, visits tothe detection volume are fast, lasting only a few microseconds, due tothe very small distance that diffusion has to carry the nucleotides. Theresult is a very low background.

Processes and systems for such real time sequencing that may be adaptedfor use with the invention are described in, for example, U.S. Pat. No.7,405,281, entitled “Fluorescent nucleotide analogs and uses therefor”,issued Jul. 29, 2008 to Xu et al.; U.S. Pat. No. 7,315,019, entitled“Arrays of optical confinements and uses thereof”, issued Jan. 1, 2008to Turner et al.; U.S. Pat. No. 7,313,308, entitled “Optical analysis ofmolecules”, issued Dec. 25, 2007 to Turner et al.; U.S. Pat. No.7,302,146, entitled “Apparatus and method for analysis of molecules”,issued Nov. 27, 2007 to Turner et al.; and U.S. Pat. No. 7,170,050,entitled “Apparatus and methods for optical analysis of molecules”,issued Jan. 30, 2007 to Turner et al.; and U.S. Pat. Pub. Nos.20080212960, entitled “Methods and systems for simultaneous real-timemonitoring of optical signals from multiple sources”, filed Oct. 26,2007 by Lundquist et al.; 20080206764, entitled “Flowcell system forsingle molecule detection”, filed Oct. 26, 2007 by Williams et al.;20080199932, entitled “Active surface coupled polymerases”, filed Oct.26, 2007 by Hanzel et al.; 20080199874, entitled “CONTROLLABLE STRANDSCISSION OF MINI CIRCLE DNA”, filed Feb. 11, 2008 by Otto et al.;20080176769, entitled “Articles having localized molecules disposedthereon and methods of producing same”, filed Oct. 26, 2007 by Rank etal.; 20080176316, entitled “Mitigation of photodamage in analyticalreactions”, filed Oct. 31, 2007 by Eid et al.; 20080176241, entitled“Mitigation of photodamage in analytical reactions”, filed Oct. 31, 2007by Eid et al.; 20080165346, entitled “Methods and systems forsimultaneous real-time monitoring of optical signals from multiplesources”, filed Oct. 26, 2007 by Lundquist et al.; 20080160531, entitled“Uniform surfaces for hybrid material substrates and methods for makingand using same”, filed Oct. 31, 2007 by Korlach; 20080157005, entitled“Methods and systems for simultaneous real-time monitoring of opticalsignals from multiple sources”, filed Oct. 26, 2007 by Lundquist et al.;20080153100, entitled “Articles having localized molecules disposedthereon and methods of producing same”, filed Oct. 31, 2007 by Rank etal.; 20080153095, entitled “CHARGE SWITCH NUCLEOTIDES”, filed Oct. 26,2007 by Williams et al.; 20080152281, entitled “Substrates, systems andmethods for analyzing materials”, filed Oct. 31, 2007 by Lundquist etal.; 20080152280, entitled “Substrates, systems and methods foranalyzing materials”, filed Oct. 31, 2007 by Lundquist et al.;20080145278, entitled “Uniform surfaces for hybrid material substratesand methods for making and using same”, filed Oct. 31, 2007 by Korlach;20080128627, entitled “SUBSTRATES, SYSTEMS AND METHODS FOR ANALYZINGMATERIALS”, filed Aug. 31, 2007 by Lundquist et al.; 20080108082,entitled “Polymerase enzymes and reagents for enhanced nucleic acidsequencing”, filed Oct. 22, 2007 by Rank et al.; 20080095488, entitled“SUBSTRATES FOR PERFORMING ANALYTICAL REACTIONS”, filed Jun. 11, 2007 byFoquet et al.; 20080080059, entitled “MODULAR OPTICAL COMPONENTS ANDSYSTEMS INCORPORATING SAME”, filed Sep. 27, 2007 by Dixon et al.;20080050747, entitled “Articles having localized molecules disposedthereon and methods of producing and using same”, filed Aug. 14, 2007 byKorlach et al.; 20080032301, entitled “Articles having localizedmolecules disposed thereon and methods of producing same”, filed Mar.29, 2007 by Rank et al.; 20080030628, entitled “Methods and systems forsimultaneous real-time monitoring of optical signals from multiplesources”, filed Feb. 9, 2007 by Lundquist et al.; 20080009007, entitled“CONTROLLED INITIATION OF PRIMER EXTENSION”, filed Jun. 15, 2007 by Lyleet al.; 20070238679, entitled “Articles having localized moleculesdisposed thereon and methods of producing same”, filed Mar. 30, 2006 byRank et al.; 20070231804, entitled “Methods, systems and compositionsfor monitoring enzyme activity and applications thereof”, filed Mar. 31,2006 by Korlach et al.; 20070206187, entitled “Methods and systems forsimultaneous real-time monitoring of optical signals from multiplesources”, filed Feb. 9, 2007 by Lundquist et al.; 20070196846, entitled“Polymerases for nucleotide analogue incorporation”, filed Dec. 21, 2006by Hanzel et al.; 20070188750, entitled “Methods and systems forsimultaneous real-time monitoring of optical signals from multiplesources”, filed Jul. 7, 2006 by Lundquist et al.; 20070161017, entitled“MITIGATION OF PHOTODAMAGE IN ANALYTICAL REACTIONS”, filed Dec. 1, 2006by Eid et al.; 20070141598, entitled “Nucleotide Compositions and UsesThereof”, filed Nov. 3, 2006 by Turner et al.; 20070134128, entitled“Uniform surfaces for hybrid material substrate and methods for makingand using same”, filed Nov. 27, 2006 by Korlach; 20070128133, entitled“Mitigation of photodamage in analytical reactions”, filed Dec. 2, 2005by Eid et al.; 20070077564, entitled “Reactive surfaces, substrates andmethods of producing same”, filed Sep. 30, 2005 by Roitman et al.;20070072196, entitled “Fluorescent nucleotide analogs and usestherefore”, filed Sep. 29, 2005 by Xu et al; and 20070036511, entitled“Methods and systems for monitoring multiple optical signals from asingle source”, filed Aug. 11, 2005 by Lundquist et al.; and Korlach etal. (2008) “Selective aluminum passivation for targeted immobilizationof single DNA polymerase molecules in zero-mode waveguidenanostructures” PNAS 105(4): 1176-81, all of which are hereinincorporated by reference in their entireties.

Subsequently, in some embodiments, the data produced comprises sequencedata from multiple barcoded DNAs. Using the known association betweenthe barcode and the source of the DNA, the data can be deconvoluted toassign sequences to the source subjects, samples, organisms, etc. Thesequences are mapped, in some embodiments, to a reference DNA sequence(e.g., a chromosome) and genotypes are assigned to the source subjects,samples, organisms, etc., e.g., by modeling, e.g., by a Hidden MarkovModel.

Some embodiments provide a processor, data storage, data transfer, andsoftware comprising instructions to assign genotypes. Some embodimentsof the technology provided herein further comprise functionalities forcollecting, storing, and/or analyzing data. For example, someembodiments comprise the use of a processor, a memory, and/or a databasefor, e.g., storing and executing instructions, analyzing data,performing calculations using the data, transforming the data, andstoring the data. In some embodiments, the processor is configured tocalculate a function of data derived from the sequences and/or genotypesdetermined. In some embodiments, the processor performs instructions insoftware configured for medical or clinical results reporting and insome embodiments the processor performs instructions in software tosupport non-clinical results reporting.

Many genotyping tests involve determining the presence or absence, ormeasuring the amount of, multiple genotypes, and an equation comprisingvariables representing the properties of multiple genotypes produces avalue that finds use in making a diagnosis or assessing the presence orqualities of a genotype. As such, in some embodiments the softwarecalculates this value and, in some embodiments, presents the value tothe user, uses the value to produce an indicator related to the result(e.g., an LED, an icon on an LCD, a sound, or the like), stores thevalue, transmits the value, or uses the value for additionalcalculations.

In some embodiments, the processor is used to initiate and/or terminatethe sequencing and data collection. In some embodiments, a device orsystem is provided comprising a user interface (e.g., a keyboard,buttons, dials, switches, and the like) for receiving user input that isused by the processor to determine one or more genotypes. In someembodiments, the device further comprises a data output for transmitting(e.g., by a wired or wireless connection) data to an externaldestination, e.g., a computer, a display, a network, and/or an externalstorage medium.

Different applications require different numbers of markers.Accordingly, the technology finds use, for example, in genotyping largepopulations wherein one may which to provide “tunable” numbers ofgenetic markers, e.g., in breeding applications (backcrossing,identification of QTL, association mapping) as well as for theprotection of IP (elite varieties) and paternity testing and forensics.In addition, embodiments of the methods find use in efficiently adding aseparate DNA barcode (an index) to each of multiple samples to provide ahigh degree of multiplexing. The technology finds use in experimentsinvolving multiple pooled environmental samples, e.g., to identify theorganisms present in multiple environments (e.g., guts from multiplehumans, different water samples, etc). The technology is useful for thequality control of biological samples (e.g., cell lines) prior toconducting expensive experiments on such samples.

In addition, the barcoding provided relates to genome sequencing.Traditionally genomes were sequenced BAC-by-BAC using Sanger technology.This approach can provide high quality assemblies for complex genomes.More recently genomes have been sequenced using a “whole genome shotgun”(WGS) approach enabled by next generation sequencing (NGS) technologies.This approach is substantially cheaper than the traditional BAC-basedordered approach, but at the cost of assembly quality. The technologyprovided herein finds use in combining the resolution of BAC-by-BACsequencing with the efficiency of NGS. For example, individual BACs aredigested and bar-coded using the technology (e.g., as embodied by theexemplary methods below) and then pooled for sequencing.Post-sequencing, each BAC is assembled individually. Overlaps among BACsare identified via sequence comparisons, thus eliminating the need togenerate a “minimum tiling path” of BACs. Sequence gaps within BACs arefilled using WGS data.

Although the disclosure herein refers to certain illustratedembodiments, it is to be understood that these embodiments are presentedby way of example and not by way of limitation.

EXAMPLES Method 1 Tunable GBS

Next-generation sequencing enables researchers to obtain large amountsof data at a reduced cost and thus provides a tremendous opportunity togenotype an individual in depth. Provided herein is a method ofrestriction enzyme digestion followed by single stranded oligonucleotideligation to barcode (e.g., index) the sources of DNAs. Two restrictionenzymes are used that generate two sites with different overhangs ateach end of each digested fragment. One site is ligated with the“barcode” oligonucleotide to permit multiplexing of samples duringsequencing. The other site is ligated with the oligonucleotide withoutthe “barcode”. The number of targeted sites is further reduced throughthe primer selection on the non-barcode site during the amplificationprocedure. See FIGS. 1 and 3.

1. Digestion

100-200 ng genomic DNA (RNase treated)

3 μl 10×NEB Buffer 4 3 μl 10×BSA 0.8 μl NspI 1 μl BfuCI

sterile H₂O to 30 μlIncubate at 37° C. for 1.5 hour

2. Ligation

After 1.5 hours incubation, add 30 μl ligation solution to genomic DNAdigestion reaction:

1.5-2.0 μl barcode oligo (e.g., from nsplxx or Nxx series) (50-100 μM)1.5-2.0 μl non-barcode oligo (e.g., bfuci12 mm, s-SEO15, etc.) (50-100μM)3 μl 10× ligase buffer1-1.5 μl T4 DNA ligasesterile water to 30 μl

Total volume per reaction is now 60 μl. Incubate for 1.5 hours at 20° C.and then at 80° C. for 20 minutes to inactivate the enzyme.

3. Purification

1) Pool all ligated samples (each 60 μl) and completely mix them evenly2) Aliquot 1.5 ml for purification, e.g., in two Qiagen columns3) Follow the manufacturer's instructions to purify the ligationproducts (eg, as provided by Qiagen, Catalog numbers 28106 or 28104).4) Elute DNA in 100 μl EB buffer4. Size selection1) Add AMPure beads to the eluted DNA according to the manufacturer'sinstructions and vortex to mix2) Incubate for 5-20 minutes at ambient temperature3) Using a magnetic particle concentrator (MPC), pellet the beadsagainst the wall of the tube.4) Remove the supernatant and wash the beads twice with 100-500 μl of70% ethanol, incubating for 30 seconds each time5) Remove all the supernatant and allow the AMPure beads to air drycompletely6) Remove the tube from the MPC, add 24-50 μl of EB, and vortex toresuspend the beads7) Using the MPC, pellet the beads against the wall of the tube oncemore and transfer the supernatant containing the purified nebulized DNAto a new microcentrifuge tube

5. Selective PCR AMPLIFICATION

1) PCR mixture:2-15 μl purified ligated DNA (approximately 100-200 ng DNA)

25 μl 2× Phusion High-Fidelity PCR Master Mix

1 μl selective primer (e.g., bfuci3ACAA, s-SEO16CTA, s-SEO16ACA) (100μM)1 μl common primer (e.g., common1, s-common1) (100 μM)sterile water to 50 μl2) Run PCR, e.g., using the PCR program: 98° C. for 30 minutes; 15cycles of 98° C. for 10 seconds, 62° C. for 30 seconds, 72° C. for 30seconds; and a final extension at 72° C. for 5 minutes.3) PCR purification according to the protocol in the QIAquick PCRpurification kit; elute with 50 μl of EB.6. Final PCR amplification

1) PCR Mixture:

5-10 μl purified selected PCR product

25 μl 2× Phusion High-Fidelity PCR Master Mix

1 μl first primer (e.g., Primer 1.1, s-SE-P1, etc.) (100 μM)1 μl second primer (e.g., final3, s-SE-P2AG, s-SE-P2GT) (100 μM)sterile water to 50 μl

The primers for the final PCR are designed to match binding sitesequences determined by the primers used in the selective PCR, e.g.,s-SE-P2AG is used in the Final PCR if s-SEO16CTA was used in theselective PCR and s-SE-P2GT is used in the Final PCR if s-SEO16ACA wasused in the selective PCR

2) Run PCR, e.g., using the PCR program of 98° C. for 30 minutes; 15cycles of 98° C. for 10 seconds, 65° C. for 30 seconds, 72° C. for 30seconds; and a final extension at 72° for 5 minutes.3) PCR purification, e.g., according to the protocol in the QIAquick PCRpurification kit; elute with 30 μl of EB.4) Measure the concentration of purified products, e.g., by nanodrop.Yield of PCR should be ≧1.0 μg5) Run Bioanalyzer DNA 1000 for size and concentration confirmation.

The sample is now ready for sequencing, data collection, and genotyping.

Method 2 Single Primer Genotyping

Provided herein is an embodiment of the technology as a method in whichrestriction enzyme digestion is followed by single-stranded oligoligation to barcode (e.g., index) the sources of DNAs. A restrictionenzyme generates an overhang at one end of each digested fragment. A“barcoded” oligonucleotide comprising a common sequence is ligated tothe digested DNAs. After ligation, the many sources of DNAs withdifferent barcoded oligonucleotides are pooled. A single primer isdesigned on a polymorphic target. In combination with the primermatching the common sequence in the barcoded oligo, this single primeris used to amplify the target site. Primers of multiple target sites canbe plexed for amplification. See FIGS. 2 and 4.

Most steps below are best performed in sterile microfuge tubes:

1. Digestion

100-200 ng genomic DNA (RNase treated)

3 μl 10×NEB Buffer 4 3 μl 10×BSA

0.8-1.2 μl restriction enzyme, e.g., NspI or BanIISterile, nuclease free H₂O to 30 μlIncubate at 37° C. for 1.5 hour

2. Ligation

After the 1.5-hour incubation, add 20 μl of ligation solution to genomicDNA digestion reaction:

1.5-2 μl barcode oligo (100 μM)2-3 μl 10× ligase buffer1-1.5 μl T4 DNA ligasesterile water to 20-22 μl

Total volume per reaction is now 50 μl. Incubate for 1.5 hours at 20° C.and then at 65° C. for 20 minutes to inactivate the enzyme. Pool allligated samples equally and completely mix them evenly.

The barcode oligo may be a single oligo or a mixture of two (or more)oligos (e.g., a oligo of the nsplxx, Nxx series).

3. Target PCR amplification1) PCR mixture:2-12 μl purified ligated DNA (approximately 100-200 ng DNA)

25 μl 2× Phusion High-Fidelity PCR Master Mix

2 μl common primer (5 μM)1.5 μl multiple target specific primers (5 μM each)sterile water to 50 μl2) Run PCR using the program; 98° C. for 30 minutes; 12 cycles of 98° C.for 10 seconds, 62° C. for 30 seconds, 72° C. for 30 seconds; and afinal extension at 72° C. for 5 minutes.3) PCR purification according to the protocol in the QIAquick PCRpurification kit; elute with 50 μl of EB.4. Final PCR amplification1) PCR mixture:2 μl 10× diluted target PCR product

25 μl 2× Phusion High-Fidelity PCR Master Mix 1 μl Illumina PCR Primer 11 μl Illumina PCR Primer 2

sterile water to 50 μl2) Run PCR using the PCR program consists of 98° C. for 30 minutes; 20cycles of 98° C. for 10 seconds, 65° C. for 30 seconds, 72° C. for 30seconds; and a final extension at 72° C. for 5 minutes.3) PCR purification according to the protocol in the QIAquick PCRpurification kit; elute with 30 μl of EB.4) Measure the concentration of purified products by nanodrop. Yield ofPCR should be ≧1.0 μg5) Run Bioanalyzer DNA 1000 for size and concentration confirmation.

The sample is now ready for sequencing, data collection, and genotyping.

In some embodiments of single primer genotyping, methods are provided asfollows:

1. Digestion

Nuclease-free water to 30 μlDNA (RNase treated) 100-200 ng

10×NEB Buffer 4 3 μl 10×BSA 3 μl

Restriction enzyme, e.g., NspI or BanII 1 μl

Total 30 μl

Incubate at 37° C. for 1.5 hours

2. Ligation

After 1.5 hour-incubation, add 30 μl ligation solution to the genomicDNA digestion reaction:

Nuclease-free water 22 μlLeft oligo (50 μM) 2 μlRight oligo (50 μM) 2 μl10× ligase buffer 3 μlT4 ligase 1 μl

Total 30 μl

Total volume per reaction is now 60 μl. Incubate for 1.5 hours at 16° C.and 80° C. for 20 minutes to inactivate the enzyme.

3. Purification

Pool all ligated samples (each 60 μl) and completely mix themAliquot 1 ml for further purification (e.g., in a Qiagen column); theremaining mixture may be retained (e.g., stored at −20° C.)Purify ligation products by suitable means, e.g., by Qiaquick PCRpurification kitElute DNA (e.g., in 100 μl EB buffer) in each tubeMeasure the concentration, e.g., by Nanodrop4. PCR amplification1) PCR mixtureMolecular grade water 13 μlPurified ligated DNA 10 μl (˜200 ng)

2× Phusion Master Mix 25 μl

First primer (e.g., s-SE-P1) 20 μM, M 1 μlSecond primer (e.g., TruSeq-final-primer) 20 μM, M 1 μl

Total 50 μl 2) Thermocycle

5. AMpure size selection (>100 by DNA enriched, 1.2:1 ratio)Add 120 μl of AMPure beads to the 100 μl eluted DNA. Vortex briefly tomixIncubate for 15 min at ambient temperatureUsing a Magnetic Particle Concentrator (MPC), pellet the beads againstthe wall of the tube.Remove the supernatant and wash the beads twice with 200 μl of 70%ethanol, incubating for 30 seconds each timeRemove the supernatant and allow the AMPure beads to air dry (˜5-10minutes)Remove the tube from the MPC, add 50 μl of EB, and vortex to resuspendthe beadsUsing the MPC, pellet the beads against the wall of the tube once moreand transfer the supernatant containing the purified nebulized DNA to anew microcentrifuge tube6. Measure the concentration of purified products by nanodrop.7. Run Bioanalyzer DNA Chip for size and concentration confirmation.8. Optional cloning of final DNA library for the confirmation

Example 1

During the development of embodiments of the technology describedherein, experiments were conducted to verify the methods provided. Asample was prepared to assess the tunable GBS method against 956previously called B73 versus Mo17 SNPs. Samples were prepared accordingto the methods provided, taking a total time of approximately one day.The methods produced 19 samples for analysis and the sequencing yielded37 million 100-bp reads, or, alternatively, approximately 2 millionreads per sample. An average of 5000 SNPs were called per sample and 820SNPs with genotyping calls for at least 12 of the 19 samples wereproduced. This resulted in a genotyping accuracy of approximately 99%.

All publications and patents mentioned in the above specification areherein incorporated by reference in their entirety for all purposes.Various modifications and variations of the described compositions,methods, and uses of the technology will be apparent to those skilled inthe art without departing from the scope and spirit of the technology asdescribed. Although the technology has been described in connection withspecific exemplary embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in relevantfields are intended to be within the scope of the following claims.

1-14. (canceled)
 15. A method for genotyping-by-sequencing a sample, themethod comprising: 1) ligating a first single-stranded oligonucleotidecomprising a first primer binding site to the first ends of nucleic acidfragments produced by digesting nucleic acids from the sample with arestriction enzyme and ligating a second oligonucleotide comprising asecond primer binding site to the second ends of the nucleic acidfragments to produce a plurality of templates; 2) amplifying templatesusing a first amplification primer that hybridizes to the first primerbinding site or complement thereof and a second amplification primerthat hybridizes to the second primer binding site or complement thereofto produce amplicons; 3) acquiring sequence reads from the amplicons;and 4) genotyping-by-sequencing the sample by determining a genotype ofthe sample using one or more sequence reads.
 16. The method of claim 15further comprising mapping one or more sequence reads to a referencesequence.
 17. The method of claim 15 wherein the nucleic acids from thesample are digested with two different restriction enzymes.
 18. Themethod of claim 15 wherein the first single-stranded oligonucleotideand/or the second oligonucleotide comprises a common sequence, a uniquesequence, and a sequence complementary to the single-stranded endproduced by the restriction enzyme.
 19. The method of claim 15 furthercomprising assembling a plurality of overlapping sequence reads.
 20. Themethod of claim 15 wherein the first amplification primer comprises: a)a 5′ portion that hybridizes to the first primer binding site orcomplement thereof; and b) a 3′ portion comprising 1-3 nucleotides, said3′ portion hybridizing to a subset of the plurality of templates. 21.The method of claim 15 comprising synthesizing or providing anamplification primer comprising a 5′ portion adjacent to a 3′ portion,the 5′ portion comprising a sequence that hybridizes to the first primerbinding site or complement thereof and the 3′ portion comprising 1 to 10nucleotides appended to the 3′ end of the 5′ portion of theamplification primer.
 22. The method of claim 15 wherein the firstsingle-stranded oligonucleotide comprises a barcode and/or the secondoligonucleotide comprises a barcode.
 23. The method of claim 22 whereinthe barcode of the first single-stranded oligonucleotide and/or thebarcode of the second oligonucleotide identifies a subject that was thesource of the nucleic acid.
 24. The method of claim 15 wherein thesecond oligonucleotide is a single-stranded oligonucleotide.
 25. Themethod of claim 15 wherein the first single-stranded oligonucleotide andthe second oligonucleotide comprise the same nucleotide sequence. 26.The method of claim 15 wherein the first single-stranded oligonucleotideand the second oligonucleotide comprise different nucleotide sequences.27. The method of claim 15 wherein: a) the first amplification primercomprises a 5′ portion and a 3′ portion, wherein: 1) the 5′ portion ofthe first amplification primer hybridizes to the first primer bindingsite or complement thereof; and 2) the 3′ portion of the firstamplification primer hybridizes to one or more bases of nucleic acidfragments, said bases being adjacent to the first primer binding site ina subset of the plurality of templates; and/or b) the secondamplification primer comprises a 5′ portion and a 3′ portion,wherein: 1) the 5′ portion of the second amplification primer hybridizesto the second primer binding site or complement thereof; and 2) the 3′portion the second amplification primer hybridizes to one or more basesof nucleic acid fragments, said bases being adjacent to the secondprimer binding site in a subset of the plurality of templates.
 28. Themethod of claim 27 wherein the 3′ portion of the first amplificationprimer and/or the 3′ portion of the second amplification primercomprises 1-10 nucleotides.
 29. The method of claim 27 wherein theamplifying produces amplicons from a subset of the plurality oftemplates, wherein each template of the subset comprises a nucleic acidfragment that comprises a portion that hybridizes to the 3′ portion ofthe first amplification primer and/or a portion that hybridizes to the3′ portion of the second amplification primer.
 30. The method of claim15 further comprising ligating a third single-stranded oligonucleotidecomprising the first primer binding site to the first ends of secondnucleic acid fragments produced by digesting nucleic acids from a secondsample with the restriction enzyme and ligating the secondoligonucleotide to the second ends of the second nucleic acid fragmentsfrom the second sample to produce a second plurality of templates;mixing the second plurality of templates with the first plurality oftemplates; genotyping-by-sequencing the first sample by determining agenotype of the first sample using one or more sequence reads, andgenotyping-by-sequencing the second sample by determining a genotype ofthe second sample using one or more sequence reads.
 31. A method forgenotyping-by-sequencing a plurality of samples, the methodcomprising: 1) ligating a first single-stranded oligonucleotidecomprising a primer binding site and a first distinct barcode to thefirst ends of first nucleic acid fragments produced by digesting nucleicacids from a first sample with a restriction enzyme and ligating acommon oligonucleotide to the second ends of the first nucleic acidfragments from the first sample to produce a first plurality oftemplates; 2) ligating a second single-stranded oligonucleotidecomprising the primer binding site and a second distinct barcode to thefirst ends of second nucleic acid fragments produced by digestingnucleic acids from a second sample with the restriction enzyme andligating a common oligonucleotide to the second ends of the secondnucleic acid fragments from the second sample to produce a secondplurality of templates; 3) amplifying templates from a template mixturecomprising the first plurality of templates mixed with the secondplurality of templates using a first amplification primer comprising a5′ portion that hybridizes to the primer binding site or complementthereof and a second amplification primer that hybridizes to the commonoligonucleotide or complement thereof to produce amplicons; 4) acquiringsequence reads from the amplicons; 5) associating sequence readscomprising a sequence of the first barcode with the first sample andassociating sequence reads comprising a sequence of the second barcodewith the second sample; and 6) genotyping-by-sequencing the first sampleby determining a genotype of the first sample using one or more sequencereads and genotyping-by-sequencing the second sample by determining agenotype of the second sample using one or more sequence reads.
 32. Themethod of claim 31 wherein the first plurality of nucleic acids from thefirst sample are from a first subject and the second plurality ofnucleic acids from the second sample are from a second subject.
 33. Acomposition comprising a nucleic acid template, said nucleic acidtemplate comprising a first single-stranded oligonucleotide ligated tothe first end of a nucleic acid fragment and a second oligonucleotideligated to the second end of the nucleic acid fragment, wherein saidfirst single-stranded oligonucleotide comprises a first primer bindingsite and said second oligonucleotide comprises a second primer bindingsite.
 34. The composition of claim 33 further comprising: 1) a firstamplification primer, said first amplification primer comprising a 5′portion and a 3′ portion, wherein the 5′ portion of the firstamplification primer hybridizes to the first primer binding site orcomplement thereof; and the 3′ portion of the first amplification primercomprises 1 to 10 nucleotides appended to the 3′ end of the 5′ portionof the amplification primer; and 2) a second amplification primer, saidsecond amplification primer comprising a 5′ portion and a 3′ portion,wherein the 5′ portion of the second amplification primer hybridizes tothe second primer binding site or complement thereof; and the 3′ portionthe second amplification primer comprises 1 to 10 nucleotides appendedto the 3′ end of the 5′ portion of the amplification primer.